Inactivation of highly transmissible livestock and avian viruses including influenza A and Newcastle disease virus for molecular diagnostics.

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection while maintaining the nucleic acid quality required for diagnostic PCR testing. This inactivation method is required to alleviate concerns about transmission potential, minimize shipping complications and cost, and enable testing in lower containment laboratories, thereby enhancing disease diagnostics through improved turn-around time. This study evaluated a panel of 10 surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial PrimeStore® molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99%, as determined by infectivity and serial passage assays. However, the detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and the shipment of infectious substances.

Virus, strain, and epitope specificities of neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB, gC, and gD, with sequence and molecular model analysis.

Three bovine monoclonal antibodies (BomAb) raised to bovine herpesvirus (BoHV) 1.1 and specific for the viral glycoproteins gB, gC, and gD were tested for reactivity to two isolates of BoHV-1.1, one of BoHV-1.2, and two of BoHV-5 in virus neutralization and indirect fluorescent antibody assays. They were also tested with other herpesviruses infecting cattle and other mammalian alphaherpesviruses, and found negative or of negligible reactivity. Their BoHV-1.1 epitope specificity was examined using competitive ELISA with peroxidase-labeled murine monoclonal antibodies (MumAb) that had been previously characterized. To explain the incongruities observed, the amino acid sequences of the epitopes and adjacent regions of BoHV-1.1, 1.2, and 5 were compared, and molecular modeling was performed using human herpesvirus 1 glycoprotein crystals as templates. The anti-gB BomAb reacted strongly with BoHV-1.1 and BoHV-1.2, and poorly or not at all with BoHV-5. It competed with a MumAb specific for a BoHV-1.1 gB epitope previously shown to only partially cross-react between BoHV-1 and BoHV-5. BoHV-5 gB has nearly identical sequence with BoHV-1.1 in the epitope region, but modeling suggested the lack of cross-reactivity of the MumAb was due to masking of the epitope in BoHV-5 by an adjacent region, which has significant sequence differences between BoHV-1.1 and BoHV-5. The BomAb reactivity could also be explained by masking, or by reactivity with the adjacent region. The anti-gC BomAb reacted strongly with one isolate of BoHV-1.1 and BoHV-1.2, less well with a heterologous isolate of BoHV-1.1, and poorly or not at all with BoHV-5. It did not compete with any of the anti-gC MumAb tested, but a target domain was suggested by BoHV-1.1, 1.2, and 5 sequence divergence. The anti-gD BomAb reacted strongly with all BoHV-1.1, 1.2, and 5 isolates tested. However, it competed with two MumAb previously shown to not cross-react between BoHV-1.1 and BoHV-5. Sequence analysis and modeling suggested the cross-reactivity of the anti-gD BomAb was due to it reacting with an epitope-adjacent region or regions conserved between BoHV-1.1 and BoHV-5, but not with other alphaherpesviruses. The results suggest the usefulness of combining in vitro biological data with sequence or structure modeling data to investigate important epitopes involved in immunity to infectious agents.

Generation by self re-fusion of bovine³ × murine² heterohybridomas secreting virus-neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB, gC, and gD.

Seventy-eight heterohybridomas (HH) stably secreting bovine monoclonal antibodies (BomAb) to Bovine herpesvirus 1 (BHV1) were produced by fusing lymph node cells from a BHV1 hyperimmunized calf with 3 types of non-secreting fusion partners. Seven were generated through fusion with the murine × murine (murine(2)) hybridoma SP2/0, 3 through fusion with bovine-murine(2) HH previously generated using cells from the same calf, and 68 through fusion with bovine(2)-murine(2) HH previously generated by sequential fusions using cells from the same calf. The chromosome number of example HH increased with increasing numbers of input fusions. A variety of indirect fluorescent antibody assay patterns was observed using the BomAb, suggesting diverse antigen specificity. Three bovine(3)-murine(2) HH secreted IgG1 BomAb neutralizing BHV1 without complement, and were chosen for further characterization. SDS-PAGE of detergent-solubilized BHV1 proteins bound to the 3 neutralizing BomAb demonstrated their individual specificities for BHV1 envelope glycoproteins gB, gC, and gD, the major neutralization targets for BHV1. The 3 HH stably secreted the BomAb in culture for over one year, and pilot-scale production of the BomAb was accomplished by in vivo and in vitro methods. A cocktail of the 3 BomAb was administered intravenously (i.v.) to a 6-month-old calf and its serum neutralization activity decreased with a half-life consistent with non-immune clearance, suggesting that BomAb may be useful for passive immune treatment of disease in cattle. Rabbits were passively protected by i.v. injection with each of the anti-gB and anti-gD BomAb when challenged i.v. with BHV1 24h later. Self re-fusion was shown to be advantageous for efficiently producing HH stably secreting host monoclonal antibodies. The BomAb described should prove useful in studies of the host immune response to BHV1, as reagents, and as sources of bovine immunoglobulin sequences.

Immunity to bovine herpesvirus 1: I. Viral lifecycle and innate immunity.

Bovine herpesvirus 1 (BHV-1) causes a variety of diseases and is globally distributed. It infects via mucosal epithelium, leading to rapid lytic replication and latent infection, primarily in sensory ganglia. Large amounts of virus can be excreted by the host on primary infection or upon recrudescence of latent infection, resulting in disease spread. The bovine immune response to BHV-1 is rapid, robust, balanced, and long-lasting. The innate immune system is the first to respond to the infection, with type I interferons (IFNs), inflammatory cytokines, killing of infected host cells, and priming of a balanced adaptive immune response. The virus possesses a variety of immune evasion strategies, including inhibition of type I IFN production, chemokine and complement binding, infection of macrophages and neutrophils, and latency. BHV-1 immune suppression contributes to the severity of its disease manifestations and to the bovine respiratory disease complex, the leading cause of cattle death loss in the USA.

Immunity to bovine herpesvirus 1: II. Adaptive immunity and vaccinology.

Bovine herpesvirus 1 (BHV-1) infection is widespread and causes a variety of diseases. Although similar in many respects to the human immune response to human herpesvirus 1, the differences in the bovine virus proteins, immune system components and strategies, physiology, and lifestyle mean the bovine immune response to BHV-1 is unique. The innate immune system initially responds to infection, and primes a balanced adaptive immune response. Cell-mediated immunity, including cytotoxic T lymphocyte killing of infected cells, is critical to recovery from infection. Humoral immunity, including neutralizing antibody and antibody-dependent cell-mediated cytotoxicity, is important to prevention or control of (re-)infection. BHV-1 immune evasion strategies include suppression of major histocompatibility complex presentation of viral antigen, helper T-cell killing, and latency. Immune suppression caused by the virus potentiates secondary infections and contributes to the costly bovine respiratory disease complex. Vaccination against BHV-1 is widely practiced. The many vaccines reported include replicating and non-replicating, conventional and genetically engineered, as well as marker and non-marker preparations. Current development focuses on delivery of major BHV-1 glycoproteins to elicit a balanced, protective immune response, while excluding serologic markers and virulence or other undesirable factors. In North America, vaccines are used to prevent or reduce clinical signs, whereas in some European Union countries marker vaccines have been employed in the eradication of BHV-1 disease.

Emerging and exotic zoonotic disease preparedness and response in the United States - coordination of the animal health component.

For the response to a zoonotic disease outbreak to be effective, animal health authorities and disease specialists must be involved. Animal health measures are commonly directed at known diseases that threaten the health of animals and impact owners. The measures have long been applied to zoonotic diseases, including tuberculosis and brucellosis, and can be applied to emerging diseases. One Health (veterinary, public, wildlife and environmental health) and all-hazards preparedness work have done much to aid interdisciplinary understanding and planning for zoonotic diseases, although further improvements are needed. Actions along the prevention, preparedness, response and recovery continuum should be considered. Prevention of outbreaks consists largely of import controls on animals and animal products and biosecurity. Preparedness includes situational awareness, research, tool acquisition, modelling, training and exercises, animal movement traceability and policy development. Response would include detection systems and specialized personnel, institutions, authorities, strategies, methods and tools, including movement control, depopulation and vaccination if available and appropriate. The specialized elements would be applied within a general (nationally standardized) system of response. Recovery steps begin with continuity of business measures during the response and are intended to restore pre-event conditions. The surveillance for novel influenza A viruses in swine and humans and the preparedness for and response to the recent influenza pandemic illustrate the cooperation possible between the animal and public health communities.